Studies On Investigation And Genetic Diversity Of Cypripedium In Changbai Mountains

The Cypripedium is the plant which is belonged to Orchidaceae and Cypripedioideae.Because of its graceful plant types,bright-coloured flowers and special flower forms are widely accepted by scientific researchers and flower amateurs.There are about 50 species and varieties in the world.There are about 36 species,1 variety and many variants in China,one of 26 are endemic species.It is reported that there are 5 species and 1 variant in JiLin province,which mainly distributed in the east areas and Changbai mountains.Changbai mountains is one of the concentrated distribution areas of wild Cypripedium in our country.What is more,there are different flower colors of Cypripedium.However,at present a lot of Cypripedium resources have not been rationally utilized,seriously destroied and theft.In recent years,the researchs about Cypripedium in Changbai mountains began to appear one after another.But it is not been reported about habitat investigation,IS SR marker and numerical taxonomy of Cypripedium in Changbai mountains.The author investigated 10 plots in Changbai mountains by consulting document and investigating plots.At the same time we investigated and collected the Cypripedium on Changbai mountains using ISSR marker and numerical taxonomy to research 72 Cypripedium materials,analyzed their genetic relationship,clearified its classification position and provided the basis for researching and preserving Cypripedium.The research results as follows:1.There are 5 species and 1 variant of Cypripedium in Changbai mountains,which were C.macranthum and its variant C.macranthum Swartz f.albiflora(Makino)Ohwi,C.calceolous,C.guttatum,C.shanxiense and C.ventricosum.Furthermore,there are many variants of Cypripedium that have not been reported.For example,labellum,Side lateral and middle speal are yellow;labellum is white,other parts are nopal,but colour of the side lateral tip become less;labellum is yellow,other parts are nopal and labellum is white,other parts are dark reddish purple;C.macranthum is the most widely distributed species.The next place is C.ventricostum,C.guttatum and C.calceolous.C.shanxiense only has been found in Jinjiang and Wangjing.Distribution areas of Cyripedium is from 41°37.29'?43°34.86'N and 125°353.31'?129°39.88'E.Vertical level of Cypripedium is at the elevation from 102.3 to 1745m.The plant is a shade species which like some of sunshine.The content of organic substance of habitat soil is 30.08?41.2mg/kg and pH is 5.20?6.22.It is suitable to grow under broad leaved forest,near the forest edge or gentle slope.Cypripedium can coexist with many vegetative plants.For example,tree layer:Q.mongolica,A.ginnala,T.amurensis and P.davidiana,brushwood layer:Vsargenti,L.bicolor,E.macropterus,field layer:C.keiskei,A.stolonifera,P.humile and so on.2.The ISSR-PCR reaction condition for Cypripedium include 20ng template DNA,1.5U Taq DNA polymerase,1.5?mol/1 primer,0.05mmol/1 dNTPs.10 ISSR primers which are suitable for Cypripedium and the optimal annealing temperature for the ISSR reaction was 50.0?55.0?.3.10 selected primers were used for ISSR amplification of the Cypripedium.A total of 73 bands were generated and average 6.7 bands were found to be polymorphic for every primer and the percentage of polymorphic band is 91.78%.4.When the similarity coefficient value was 0.66,the 72 materials of Cypripedium were divided into 4 groups.The first group included 26 materials of C.macranthum,8 materials of C.macranthum Swartz f.albiflora(Makino)Ohwi and 6 variants of Cypripedium,The second group included 8 materials of C.ventricosum.C.guttatum was separately clustered into the fourth group and another 16 resources were clustered into the third group,which included C.calceolous and C.shanxiense.5.The genetic relationships of the Cypripedium have been analyzed using software by Q cluste and R cluster.Q cluster analysis shows that 72 materials of Cypripedium were divided into 3 groups.The analysis of the R clustering indicated that 26 characters were important in the classification of Cypripedium.6.From the ISSR marker results and the ornamental character quantity classification results of the Cypripedium,we can preliminarily think that:there is a certain similarity,but genetic relationshiptries of the Cypripedium has not close relation with the morphological feature.It shows high genetic diversity among all materials.